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The Controller

The Controller is an integrated genetic circuit, comprised of two plasmids, that directs the copy number and transcription of the primary devices in our system.

Introduction

The Bactoblood organism needs to exist in two different states: one form that is genetically stable and able to grow under normal laboratory conditions, and a second state that is highly differentiated, unable to grow, and devoid of genetic material. To bring about the transformation to the differentiated state, we needed a controller that could be easily triggered by an external cue. This controller required a large dynamic range between the off and on states, the ability to maintain and overexpress a large number of genes, and ideally employed a low-cost inducer. Therefore, we designed a controller based on a two plasmid system. One plasmid stably replicates the various biosynthetic operons of our system at single copy in a transcriptionally-inactive state. The second plasmid houses the genes necessary for activation of the operon plasmid. When activated with iron, the copy number of the operon plasmid increases to high-copy and the transcription of the operons is activated.

Design and construction

We designed a two-plasmid architecture in which the biosynthetic operons reside on a single-copy bacterial artificial chromosome (BAC). The operons are under the transcriptional control of T7 promoters of various strengths. The BAC also contains an R6K origin of replication. In most strains of E. coli, this origin is silent as it requires the expression of the pir gene for replication. The second plasmid in our controller is a low-copy pSC101-derived plasmid that houses the T7 RNA polymerase and pir genes under the control of an iron-inducible promoter.

Construction of an iron-responsive PoPS-generating device

To construct this system, first we needed a promoter that was induced by iron. Microarray studies suggested that the yfbE promoter of E. coli might function as an iron-responsive PoPS-generating device. We therefore constructed a Biobrick derived from the yfbE promoter and constructed an RFP reporter composite part derived from this basic part. We examined the fluorescence of cells harboring this part both as a function of external iron concentration and growth phase. The yfbE promoter part had the ideal qualities for our controller: it is induced 100-fold as the bacteria emerge from the mid-log phase of growth, but only in the presence of exogenous iron.

Vectorology of the iron promoter characterization construct

An iron-inducible promoter(To the right): Cells were transformed with an RFP transcriptional reporter device derived from our yfbE promoter part and grown with or without exogenous iron to various densities and then analyzed for fluorescence by cytometry.

Construction of an iron-dependent transcription device

To control gene expression we needed to place the T7 RNA polymerase under the control of the yfbE promoter on a pSC101-derived plasmid. We therefore made a T7 RNA polymerase basic part and constructed a library of composite parts containing the yfbE part, one of nine ribosome binding site parts of different strengths, and the T7 RNA polymerase gene with a GTG or an ATG start codon. We constructed these composite parts in the pSC101 Biobrick plasmid I716101 and then examined their activity in an engineered E. coli strain, GH455G, containing a genome-integrated cassette with GFP under the control of a T7 promoter. Of the composite parts we constructed, only the composite part with the weakest ribosome binding site and a GTG start codon showed iron-dependent GFP production. All composite parts with an ATG start were too active and toxic, while the other ribosome binding sites were either constitutively on or off.

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 <div align="center">
    <p><a href="https://2007.igem.org/Berkeley_UC">&lt;&lt;&lt; Return to UC Berkeley iGEM 2007 </a></p>
-   <p> <a href="https://2007.igem.org/BerkiGEM2007Present1">&lt;&lt;Previous Section: Oxygen Transport</a> | <a href="https://2007.igem.org/BerkiGEM2007Present3">Next Section: Controller&gt;&gt;</a></p>
+   <p> <a href="https://2007.igem.org/BerkiGEM2007Present4">&lt;&lt;Previous Section:  Chassis</a> | <a href="https://2007.igem.org/BerkiGEM2007Present5">Next Section: Genetic Self-Destruct&gt;&gt;</a></p>
 </div>
-<h1 align="center">The Chassis</h1>
+<h1 align="center">The Controller</h1>
-<br />
-<p align="justify">There are several core issues associated with introducing <em>E. coli</em> into the bloodstream of human beings or other animals.  First is the issue of sepsis. <em>E. coli</em> possesses a species called lipid X, or endotoxin, in its outer membrane  which causes the release of TNFalpha in humans. This is an essential  process of the innate immune system, but high doses of lipid X can be  lethal. Bactoblood must have some ability to avoid this series of  events. Similarly, there are a variety of additional features in <em>E. coli</em> that can elicit strong adaptive immune responses including the pili and  flagella. From the bacterium's perspective, the interaction with the  bloodstream is no more desirable. The complement system, another core  component of the innate immune response, can kill bacteria directly.  Additionally, phagocytic cells including macrophages and neutrophils,  can engulf and kill <em>E. coli</em>.  Fortunately for our purposes, there are a variety of modifications we can make to circumvent these problems.</p>
-<h2 align="center">The <em>E. coli</em> Outer Surface</h2>
-<p align="justify"><img src="https://static.igem.org/mediawiki/2007/e/e4/BerkiGEM2007-ColiOuterSurface.gif" alt="" width="699" height="475" align="right">To understand these modifications, we must first understand what features are present in <em>E. coli</em> strain MC1061, our starting point for Bactoblood.  Like most strains of <em>E. coli</em> used in the lab, MC1061 comes from the MG1655 lineage and is a "rough"  strain. Unlike other "smooth" strains, MC1061 lacks surface-displayed  capsular polysaccharides known as K capsules and O antigens. It retains  the general 2-membrane architecture present in gram-negative bacteria.  In between these membranes is the periplasmic space which is composed  of a gel-like carbohydrate-rich polymer called peptidoglycan. The inner  membrane is composed of a lipid bilayer and a variety of proteins. The  outer membrane similarly is a lipid bilayer, and the lipid component of  it is called lipopolysaccharide, or LPS. The structure of LPS at it's  core is a 6 fatty acid lipid called lipid X. When O antigen polymer  chains are present, they are covalently attached to the outer leaf of  LPS. K capsules are similarly embedded in the outer leaf of the outer  membrane, but they are not directly attached to LPS. Other components  of the outer membrane include a structural protein, LPP, and a variety  of other proteins. This outer surface is the critical region of the  bacterium for understanding how it interacts physically with the  outside world. When the bloodstream "looks" at E. coli, what it "sees"  is the outer membrane because everything else is stuck inside.  Modifications such as O antigens and K capsules therefore have dramatic  effects on the bacterium's interactions with the outside world.</p>
-<p align="justify">&nbsp;</p>
-<h2 align="center">Capsular Polysaccharides</h2>
-<p align="center"><img src="https://static.igem.org/mediawiki/2007/e/e4/BerkiGEM2007_O16andK1Clusters.jpg" width="893" height="117" alt=""></p>
-<p align="justify"> The carbohydrates embedded in the outer membrane are extremely diverse within the <em>E. coli</em> species. Both K capsules and O antiens are linear carbohydrates  polymer, but at least 150 chemically-distinct O antigens exist in one <em>E. coli</em> strain or another. Similarly, at least 100 chemically-distinct K  capsules have been described. Almost all pathogenic strains of <em>E. coli</em> have some sort of capsular polysaccharide and are referred to as  "smooth" strains. The rough vs. smooth distinction refers to a visibly  discernible quality of their colonies. The particular choice of  carbohydrate present in a bacterium is essential to its ability to  survive in its living environment. For pathogenic and commensal  bacteria, specific O or K carbohydrates are appropriate for distinct  areas of the body (blood stream, urinary tract, intestines) and also  for distinct animal types (birds, pigs, humans, cows, etc.). Over 90%  of human cases of <em>E. coli</em> bacteremia (the clinical word for  having bacteria in the bloodstream) are caused by strains that have a  specific type of K capsule called K1. K1 is a long linear polymer of  sialic acid that extends about half the diameter of the bacterium  beyond its surface. Because polysialic acid is a frequent coating on  mammalian cells, the human immune system does not recognize K1 as  foreign. Bacteria with a K1 capsule are therefore resistant to both  innate and adaptive immune responses. Proper display of a K1 capsule  requires the concomitant expression of any of several O antigens. For  our studies, we have chosen O16. Genetically, the K1 capsule requires  14 genes encoded within a 20kb cassette. The O16 antigen requires 11  genes encoded within a 12kb cassette. Together, these surface  modifications allow the bacterium to avoid detection by the immune  system and should extend the serum half-life of Bactoblood to several  hours rather than the less-than-5 minutes observed with rough strains.  Both of these gene clusters have been installed into the genome of  MC1061 in the course of preparing our chassis strain, MC828U.</p>
-<p align="justify">&nbsp;</p>
-<h2 align="center">Lipid X and Its Variants</h2>
 <div align="justify">
-   <p>The lipid X component of the LPS in <em>E. coli</em> contains 6 acyl  chains. Mammalian blood contains a protein called LBP that scavenges  this molecule from both live and lysed bacteria and transfers it to  toll-like receptor 4 present on mammalian cells. These events initiate  a signal transduction cascade resulting in the release of a protein  called TNFalpha. The inflammatory response to these events at low doses  helps your body fight off bacterial infections. At higher doses, it can  result in organ failure and even death. The lipid X moiety present in a  variety of other bacteria do not initiate this cascade of events.  Similarly, a pentaacylated variant of the <em>E. coli</em> lipid X is  1000x less agonistic of this response. Our bacteria synthesize this  pentaacylated variant due to the deletion of the gene responsible for  attaching the sixth acyl chain, <em>msbB</em>.</p>
+   <p>The Controller is an integrated genetic circuit, comprised of two  plasmids, that directs the copy number and transcription of the primary  devices in our system.<br />
-   <p>&nbsp;  </p>
+   </p>
 </div>
-<h2 align="center">Additional Cell-Surface Epitopes</h2>
+<h2 align="center">Introduction</h2>
-<div align="justify">
+<p align="justify">The Bactoblood organism needs to exist in two different states: one  form that is genetically stable and able to grow under normal  laboratory conditions, and a second state that is highly  differentiated, unable to grow, and devoid of genetic material. To  bring about the transformation to the differentiated state, we needed a  controller that could be easily triggered by an external cue. This  controller required a large dynamic range between the off and on  states, the ability to maintain and overexpress a large number of  genes, and ideally employed a low-cost inducer. Therefore, we designed  a controller based on a two plasmid system. One plasmid stably  replicates the various biosynthetic operons of our system at single  copy in a transcriptionally-inactive state. The second plasmid houses  the genes necessary for activation of the operon plasmid. When  activated with iron, the copy number of the operon plasmid increases to  high-copy and the transcription of the operons is activated.</p>
-  <p>Essentially any component on the surface of the bacteria has the  potential to elicit either innate or adaptive immune responses. Of  those present on MC1061's surface, type I pili and flagella are known  to elicit such responses. Each of these features is encoded within  multi-gene operons encoding protein assemblies that extend out from the  bacteral surface. Type I pili allow bacteria to adhere to the surface  of mammalian cells. Flagella are the "propellers" that allow the  bacteria to swim during chemotaxis. Bactoblood does not require either  of these activities, so these genes were deleted in the chassis strain. </p>
+<h2 align="center">Design and construction</h2>
-  <p>&nbsp;</p>
+<p align="justify">We designed a two-plasmid architecture in which the biosynthetic  operons reside on a single-copy bacterial artificial chromosome (BAC).  The operons are under the transcriptional control of T7 promoters of  various strengths. The BAC also contains an R6K origin of replication.  In most strains of <em>E. coli</em>, this origin is silent as it requires the expression of the <em>pir</em> gene for replication. The second plasmid in our controller is a  low-copy pSC101-derived plasmid that houses the T7 RNA polymerase and <em>pir</em> genes under the control of an iron-inducible promoter.</p>
-</div>
+<p><img src="https://static.igem.org/mediawiki/2007/5/5e/Berk-DT_Figure_1.png" alt="" width="642" height="574" align="left"></p>
-<h2 align="center">Growth Control by Iron Restriction</h2>
-<div align="justify">
-  <p>A critical challenge in the development of Bactoblood is the ability to  safely inject a large quantity of bacteria into the bloodstream. It is  essential that these bacteria do not grow for a safe administration. We  have adopted a two-tier strategy for eliminating the possibility of  growth. First, we make our bacteria unable to grow in the bloodstream  due to their inability to acquire a specific nutrient, iron. Secondly,  we introduce a genetic kill switch device that destroys the bacterium's  DNA once it has produced all the necessary biochemical components  needed to carry oxygen. <em>E. coli</em> can acquire iron by either  high-affinity or low-affinity iron transport. When they grow in LB  media, they use low-affinity transport which involves specific  membrane-embedded transporters that can pump in free iron ions. In  animal body fluids, the concentration of these ions is less than 10-15  molar. The iron is sequestered in small molecule chelators such as heme  or proteins like transferrin and ferritin. Acquiring iron in such an  environment requires more elaborate processes involving secreted  chelators that can grab the iron from these proteins. These processes,  collectively referred to as high-affinity iron transport, all require  the function of <em>tonB</em>.  By deleting <em>tonB</em> in our chassis  organism, we obtain a strain that retains its ability to grow normally  in LB media but is unable to grow in mammalian body fluids.</p>
-  <p>&nbsp;  </p>
-</div>
-<h2 align="center">Characterization of the Chassis, MC828U</h2>
-<p align="justify">The genotype of our chassis organism is: </p>
-<div align="justify">
-  <pre><strong>MC828U</strong> delta(<em>araA</em>-<em>leu</em>)7697 <em>araD139</em> delta(<em>codB</em>-<em>lac</em>)=delta<em>lac74</em> <em>galK16</em>   <em>
-galE15</em> <em>mcrA0</em> <em>relA1</em> <em>rpsL150</em> <em>spoT1</em> <em>mcrB9999</em> <em>hsdR2</em> O16(delta<em>wbbL</em>)
-K1(delta<em>neuS</em>) delta<em>msbB</em> delta<em>fim</em> delta<em>tonB</em> delta<em>flhCD</em>   <em>
-upp</em>::(Ptet-<em>wbbL</em>-<em>neuS</em>)  </pre>
-</div>
-<p align="justify">To illustrate the function of our chassis, here we show the function  of 2 critical features of chassis: its ability to survive in serum and  loss of chemotaxis due to the deletion of flagella. </p>
-<p align="justify"><br>
-  The serum assay shows the chassis' ability to survive in serum due to its K1:O16 additions and msbB deletion.</p>
-<p><img src="https://static.igem.org/mediawiki/2007/9/9c/Serumassaypic.jpg" alt="" width="835" height="685" align="middle"></p>
 <p>&nbsp;</p>
-<p>In a swarming assay done to demonstrate the effect of flagella  deletion, the wildtype MC1061 cells on the left plate were able to swim  out much farther than those of the MC828U chassis, shown on the right  plate.</p>
-<p><br>
-    <img src="https://static.igem.org/mediawiki/2007/f/f3/SwarmingPic.jpg" alt="" width="492" height="272" align="absmiddle"></p>
 <p>&nbsp;</p>
-<p align="center"><a href="https://2007.igem.org/Berkeley_UC">&lt;&lt;&lt; Return to UC Berkeley iGEM 2007 </a></p>
+<p><img name="" src="https://static.igem.org/mediawiki/2007/d/d3/Berk-DT_Figure_Legend.png" width="384" height="323" alt=""></p>
-<p align="center"> <a href="https://2007.igem.org/BerkiGEM2007Present1">&lt;&lt;Previous Section: Oxygen Transport</a> | <a href="https://2007.igem.org/BerkiGEM2007Present3">Next Section: Controller&gt;&gt;</a></p>
+<h4>&nbsp;</h4>
+<h4>&nbsp;</h4>
+<h4>&nbsp;</h4>
+<h4>&nbsp;</h4>
+<h4>&nbsp;</h4>
+<p><img src="https://static.igem.org/mediawiki/2007/0/0a/BerkiGEM2007-yfbEcytometry.jpg" alt="" name="" width="653" height="937" align="right"></p>
+<h2 align="center">Construction of an iron-responsive PoPS-generating device</h2>
+<p align="justify">To construct this system, first we needed a promoter that was induced by iron.  Microarray studies suggested that the <em>yfbE</em> promoter of <em>E. coli</em> might function as an iron-responsive PoPS-generating device.  We therefore constructed a Biobrick derived from the <em>yfbE</em> promoter and constructed an RFP reporter composite part derived from  this basic part. We examined the fluorescence of cells harboring this  part both as a function of external iron concentration and growth  phase. The <em>yfbE</em> promoter part had the ideal qualities for our  controller: it is induced 100-fold as the bacteria emerge from the  mid-log phase of growth, but only in the presence of exogenous iron.</p>
+<p align="justify">&nbsp;</p>
+<p align="center"><strong>Vectorology of the iron promoter characterization construct</strong></p>
+<p align="center"><img src="https://static.igem.org/mediawiki/2007/2/2a/BerkiGEM2007-Figure-Piron-vector.png" alt="" name="" width="295" height="221" align="texttop"></p>
+<h4>&nbsp;</h4>
+<h4>&nbsp;</h4>
+<h4>&nbsp;</h4>
+<h4>&nbsp;</h4>
+<p>&nbsp;</p>
+<p>&nbsp;</p>
+<p>&nbsp;</p>
+<p><strong>An iron-inducible promoter(To the right)</strong>: Cells were transformed with an RFP  transcriptional reporter device derived from our yfbE promoter part and  grown with or without exogenous iron to various densities and then  analyzed for fluorescence by cytometry.</p>
+<p>&nbsp;</p>
+<p align="justify">&nbsp;</p>
+<p>&nbsp;</p>
+<h2>Construction of an iron-dependent transcription device</h2>
+<div align="justify">To control gene expression we needed to place the T7 RNA polymerase  under the control of the yfbE promoter on a pSC101-derived plasmid. We  therefore made a T7 RNA polymerase basic part and constructed a library  of composite parts containing the yfbE part, one of nine ribosome  binding site parts of different strengths, and the T7 RNA polymerase  gene with a GTG or an ATG start codon. We constructed these composite  parts in the pSC101 Biobrick plasmid I716101 and then examined their  activity in an engineered <em>E. coli</em> strain, GH455G, containing a  genome-integrated cassette with GFP under the control of a T7 promoter.  Of the composite parts we constructed, only the composite part with the  weakest ribosome binding site and a GTG start codon showed  iron-dependent GFP production. All composite parts with an ATG start  were too active and toxic, while the other ribosome binding sites were  either constitutively on or off.
+</div>
+<h2>Construction of an iron-dependent copy number device</h2>
+<h2>&nbsp;</h2>
+<h2>Conclusion</h2>
+<p>&nbsp;</p>
+<p align="center">&nbsp;</p>
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